Tumor progression in diploid cells is a phenomenon that is characterized by a complex, usually slowly developing sequence of cellular reactions that is entrained by a rapid initiating event and is culminated by the development of a tumor ("cancer"). Initiation is necessary, but not sufficient to cause cells to acquire the ability to form tumors. Separating the appearance of malignant tumours from the initiating even are a series of apparently sequential phenotypic alterations in the population of cells at risk. Tumor progression has been viewed as a biological phenomenon essential to neoplastic transformation, during which initiated cells progressively develop phenotypic modifications that allow their selective multiplication and facilitate the emergence of successively more aberrant cells. Despite the general occurrence of this phenomenon of phenotypic alteration during carcinogenesis, the roles of the various phenotypically distinct populations of cells in emergence of tumorigenic cells is not clear. The goal of this project is to elucidate the role in tumor progression of cell populations expressing variant phenotypes. Sepcifically, we aim to determine whether preneoplastic phenotypes are obligatory or coincidental to tumor development, and, if obligatory, to determine whether various phenotypes represent linked sequential stages in the development of cancer. This project uses a line of cultured diploid hepatic epithelial cells from inbred rats that express selected hepatic function and produce carcinomas following transplantation into isogeneic hosts after in vitro treatment with MNNG. Tumor progression in cell populations in vitro is slow and is associated with the expression of several reproducible phenotypic changes which appear to be progressive and, possibly, sequential. Cultured cells can be mainpulated as single cells, and should allow the determination of whether any or all of the subpopulations that express altered phenotypes are required precursors of tumorigenic cells.